Abstract

North American monarch butterflies (Danaus plexippus) undergo a spectacular fall migration. In contrast to summer butterflies, migrants are juvenile hormone (JH) deficient, which leads to reproductive diapause and increased longevity. Migrants also utilize time-compensated sun compass orientation to help them navigate to their overwintering grounds. Here, we describe a brain expressed sequence tag (EST) resource to identify genes involved in migratory behaviors. A brain EST library was constructed from summer and migrating butterflies. Of 9,484 unique sequences, 6068 had positive hits with the non-redundant protein database; the EST database likely represents approximately 52% of the gene-encoding potential of the monarch genome. The brain transcriptome was cataloged using Gene Ontology and compared to Drosophila. Monarch genes were well represented, including those implicated in behavior. Three genes involved in increased JH activity (allatotropin, juvenile hormone acid methyltransfersase, and takeout) were upregulated in summer butterflies, compared to migrants. The locomotion-relevant turtle gene was marginally upregulated in migrants, while the foraging and single-minded genes were not differentially regulated. Many of the genes important for the monarch circadian clock mechanism (involved in sun compass orientation) were in the EST resource, including the newly identified cryptochrome 2. The EST database also revealed a novel Na+/K+ ATPase allele predicted to be more resistant to the toxic effects of milkweed than that reported previously. Potential genetic markers were identified from 3,486 EST contigs and included 1599 double-hit single nucleotide polymorphisms (SNPs) and 98 microsatellite polymorphisms. These data provide a template of the brain transcriptome for the monarch butterfly. Our "snap-shot" analysis of the differential regulation of candidate genes between summer and migratory butterflies suggests that unbiased, comprehensive transcriptional profiling will inform the molecular basis of migration. The identified SNPs and microsatellite polymorphisms can be used as genetic markers to address questions of population and subspecies structure.

A. Sequencing the monarch brain cDNA clones and assembly into contigs. B. Annotating the monarch EST database (described in text). C. The 6068 ESTs annotated against the non-redundant protein database are represented in the pie-chart according to the best matching sequences.

Expression profiles of selected genes between summer and migratory butterflies.

Relative expression of the mRNA levels of allatotropin, juvenile hormone acid methyltransferase (JHAMT), takeout, juvenile hormone (JH) epoxide hydrolase, foraging, single-minded, and turtle were examined by qPCR. The analysis was performed on RNA from summer monarchs (three 12-animals sets of head RNA collected during summer 2005) and on RNA from migratory monarchs (three 12-animal sets of head RNA collected during fall 2005, and three 12-animals sets of head RNA collected during fall 2006). Only the 2005 RNA from migratory butterflies was used for analysis of single-minded and turtle gene expression. The results were normalized with rp49 and then averaged. The average level of each gene in the migrants was normalized to 1.0 for graphing. *** p<0.001, ** p<0.01, * p<0.05

A. Electropherogram of valine codon in the BF01056B1B12.fl EST. B. Partial sequence of the Na+/K+ ATPase from the monarch, queen butterfly (Danaus gilippus), and sheep. H1 and H2 are transmembrane domains. The residues in bold in the sheep sequence have been shown by mutagenesis to confer ouabain resistance when mutated, and positions 111 and 122 are indicated [35]. The Monarch-EST sequence is from two EST clones (BF01056B1B12.fl & BF01017B2C11.fl) from the monarch EST database. The Monarch-Previous sequence is the previously reported monarch sequence which has an amino acid change at one critical site (shown in red) [32], [34]. Both of the EST sequences have a second amino acid change at a second critical site (shown in green). The queen butterfly sequence has neither change [34].